Longitudinal metabolic and gut bacterial profiling of pregnant women with previous bariatric surgery
Kiana A. West
2019-09-24
- Figure S1: Summary of sampling by individual
- Figures S2 and S5: Overlayed median 1H NMR spectra
- Figure 1: Longitudinal modelling of metabolite concentrations
- Table S5: Maternal metabolic changes depend on type of bariatric surgery
- Figure S3: Maternal metabolic changes depend on type of bariatric surgery
- Figure S7: Relative concentrations of urinary host-microbial co-metabolites vary depending on bariatric surgery sub-type
- Figure 2: Gut bacterial alterations in malabsorptive patients
- Table S7: Differentially abundant amplicon sequence variants
- Figure 3: Integration of serum, urine and faecal profiles
- Figure 4: Relative concentrations of metabolites correlate with maternal insulin resistance and fetal/birth weight
- Table S8: Relative concentrations of metabolites correlate with maternal insulin resistance and fetal/birth weight
- Figure S8: Key metabolites are not associated with percentage of weight lost or length of time since surgery in malabsorptive patients
All code used to generate results within the manuscript and the supplementary files is contained here
- For all P values, an alpha of 0.05 was used
- The Benjamini-Hochberg method was used to control for false discovery rate
Parameters
save_dir <- "output" # where to save figures and tables
if (!dir.exists(save_dir)) { stop(paste("save_dir", save_dir, "does not exist")) }
color_pal <- list(NBS = "purple", RES = "coral2", MAL = "darkturquoise")Load data
load("PBS_data.Rdata")
ls()## [1] "color_pal" "integrals" "med_specs_serum" "med_specs_urine"
## [5] "pkgs" "ps_M" "rdp.species" "save_dir"
## [9] "silva.species"Figure S1: Summary of sampling by individual
Contribution of samples by study group at each time point
mb <- data.frame(Patient.TP = sample_names(ps_M),
faeces = TRUE)
# order individuals by number of time points where samples were collected
ord <- table(integrals$Study.no) %>%
sort(decreasing = TRUE) %>%
names()
# which samples were collected for each individual at each time point
all_samples <- integrals %>%
filter(Time_point != 7) %>%
mutate(urine = ifelse(!is.na(PAG), TRUE, NA),
serum = ifelse(!is.na(isoleucine), TRUE, NA)) %>%
full_join(mb, by = "Patient.TP") %>%
mutate(Sampling = case_when(urine == TRUE & serum == TRUE & faeces == TRUE ~ "Serum/Urine/Faeces",
urine == TRUE & serum == TRUE & is.na(faeces) ~ "Serum/Urine",
urine == TRUE & is.na(serum) & faeces == TRUE ~ "Urine/Faeces",
is.na(urine) & serum == TRUE & faeces == TRUE ~ "Serum/Faeces",
urine == TRUE & is.na(serum) & is.na(faeces) ~ "Urine only",
is.na(urine) & serum == TRUE & is.na(faeces) ~ "Serum only"),
Sampling = factor(Sampling, levels = c("Serum/Urine/Faeces", "Serum/Urine", "Urine/Faeces",
"Serum/Faeces", "Urine only", "Serum only")),
Study.no = factor(Study.no, levels = ord))Make plot
p <- ggscatter(all_samples,
x = "Study.no",
xlab = "Individual",
y = "Time_point_label",
ylab = "Time point",
color = "Sampling",
palette = c("chartreuse3", "mediumorchid3", "dodgerblue",
"darkorange", "gold2", "red3"),
size = "Obesity") +
theme(axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
legend.spacing.x = unit(2, "mm"),
legend.title = element_blank()) +
grids(linetype = "dashed", axis = "x", color = "grey")
p <- facet(p, facet.by = "Group", scales = "free", ncol = 1)
Figures S2 and S5: Overlayed median 1H NMR spectra
Serum (Figure S2) and urine (Figure S5) median spectra were overlayed for the three study groups
plot_med_specs <- function(to_plot, save_dir, save_name) {
p <- plot_ly(to_plot, x = ~ppm, y = ~MAL, name = "MAL", type = "scatter", mode = "lines", alpha = 0.8, color = color_pal$MAL) %>%
add_trace(y = ~RES, name = "RES", mode = "lines", alpha = 0.8, color = color_pal$RES) %>%
add_trace(y = ~NBS, name = "NBS", mode = "lines", alpha = 0.8, color = color_pal$NBS) %>%
layout(xaxis = list(autorange = "reversed", size = 18),
yaxis = list(title = "Integral (a.u.)", size = 18))
htmlwidgets::saveWidget(p, file = file.path(getwd(), save_dir, save_name))
}
# serum
plot_med_specs(med_specs_serum, save_dir, "figureS2_median_spectra_serum.html")
# urine
plot_med_specs(med_specs_urine, save_dir, "figureS5_median_spectra_urine.html")Figure S2 (annotated) 
Figure S5 (annotated) 
Key: 1 leucine; 2 isoleucine; 3 isobutyrate; 4 D-β-hydroxybutyrate ; 5 N- acetyl glycoprotein (GlycA) ; 6 glutamine ; 7 lipids (C=CCH2C=C) ; 8 unsaturated lipids (CH=CH); 9 valine; 10 α-ketoisovalerate; 11 methylmalonate; 12 creatinine; 13 p-cresol sulfate; 14 p-hydroxyphenylacetate; 15 phenylacetylglutamine; 16 unknown; 17 indoxyl sulfate
Figure 1: Longitudinal modelling of metabolite concentrations
Metabolites were considered discriminatory (malabsorptive vs control) if their NMR peaks were correlated (>=0.45) with the predictive component of a valid OPLS-DA model at any time point. A representative peak for each metabolite was integrated to calculate relative concentration.
The behavior of key discriminatory metabolites over time was assessed and compared between control and malabsorptive groups using santaR time-series analysis.
Abbreviations: PAG: phenylacetylglutamine, PCS: p-cresol sulfate, IS: indoxyl sulfate, PHPA: p-hydroxyphenylacetate, unkn: unknown, aKIV: α-ketoisovalerate, MM: methylmalonate, THB: D-β-hydroxybutyrate, NAGP: N-acetyl glycoprotein
################## DEFINED VARIABLES ##################
# remove restrictive group
input_data <- subset(integrals, integrals$Group != "Restrictive" & integrals$Time_point != 7)
# column names
metab_names <- c("leucine", "isoleucine", "isobutyrate", "NAGP", "glutamine", "THB")
tp_col <- "Time_point"
patient_col <- "Study.no"
group_col <- "Group"
tp_labs <- c("T1", "T2", "T3", "T4", "T5", "T6")
plot_titles <- c("Serum - Leucine",
"Serum - Isoleucine",
"Serum - Isobutyrate",
"Serum - GlycA",
"Serum - Glutamine",
"Serum - D-beta-hydroxubutyrate")
# see santaR vignette for choosing this value
degrees_freedom <- 5
# add custom locations (y axis) on plots for p values
pval_locs = c(27, 20, 2.1, 5.0, 8.0, 17)
###########################################################Serum metabolites
# run santaR time series analysis
sp <- santaR_auto_fit(inputData = input_data[,metab_names], ind = input_data[[patient_col]],
time = as.numeric(input_data[[tp_col]]), group = input_data[[group_col]],
df = degrees_freedom)
# generate a plot for each metabolite
for (metab in 1:length(metab_names)) {
p <- santaR_plot(sp[[metab_names[metab]]],
showIndPoint=FALSE,
showIndCurve=FALSE,
xlab = NULL,
ylab = NULL,
colorVect = unlist(color_pal[-2]),
title = plot_titles[metab]) +
scale_x_continuous(breaks = 1:length(tp_labs),
labels = tp_labs) +
theme(plot.title = element_text(size = 6),
legend.position = "None",
axis.text = element_text(size = 5),
panel.grid.major = element_blank(),
panel.grid.minor = element_blank()) +
# add p value
annotate("text",
size = 2,
x = 5,
y = pval_locs[metab],
label = paste("P = ", round(sp[[metab_names[metab]]]$general$pval.dist, digits = 4)))
assign(metab_names[metab], p)
}Urine metabolites
################## DEFINED VARIABLES ##################
# remove restrictive group
input_data <- subset(integrals, integrals$Group != "Restrictive" & integrals$Time_point != 7)
# column names
metab_names <- c("PAG", "PCS", "IS", "PHPA", "unkn", "valine", "aKIV", "MM", "creatinine")
tp_col <- "Time_point"
patient_col <- "Study.no"
group_col <- "Group"
tp_labs <- c("T1", "T2", "T3", "T4", "T5", "T6")
plot_titles <- c("Urine - PAG",
"Urine - PCS",
"Urine - IS",
"Urine - PHPA",
"Urine - Unknown",
"Urine - Valine",
"Urine - alpha-Ketoisovalerate",
"Urine - Methylmalonate",
"Urine - Creatinine")
# see santaR vignette for choosing this value
degrees_freedom <- 5
# add custom locations (y axis) on plots for p values
pval_locs = c(28, 75, 6.5, 6.5, 13, 12, 22, 12, 550)
############################################################ run santaR time series analysis
sp <- santaR_auto_fit(inputData = input_data[,metab_names], ind = input_data[[patient_col]],
time = as.numeric(input_data[[tp_col]]), group = input_data[[group_col]],
df = degrees_freedom)
# generate a plot for each metabolite
for (metab in 1:length(metab_names)) {
p <- santaR_plot(sp[[metab_names[metab]]],
showIndPoint=FALSE,
showIndCurve=FALSE,
xlab = NULL,
ylab = NULL,
colorVect = unlist(color_pal[-2]),
title = plot_titles[metab]) +
scale_x_continuous(breaks = 1:length(tp_labs),
labels = tp_labs) +
theme(plot.title = element_text(size = 6),
legend.position = "None",
axis.text = element_text(size = 5),
panel.grid.major = element_blank(),
panel.grid.minor = element_blank()) +
# add p value
annotate("text",
size = 2,
x = 5,
y = pval_locs[metab],
label = paste("P = ", round(sp[[metab_names[metab]]]$general$pval.dist, digits = 4)))
assign(metab_names[metab], p)
}(1b) Relative concentration of PAG was also higher in neonatal urine from malabsorptive group.
pb <- ggboxplot(integrals,
x = "Group_label",
y = "PAG_B",
color = "Group_label",
ylim = c(-1,11),
add = "jitter",
size = 0.25,
add.params = list(size = 0.5),
palette = unlist(color_pal),
legend = "none",
xlab = FALSE,
ylab = "Integral (a.u.)",
title = "Neonatal Urine - PAG") +
theme(axis.text=element_text(size = 5),
axis.title = element_text(size = 6),
plot.title = element_text(size = 8, hjust = 0.5)) +
# add significance
stat_compare_means(comparisons = list(c("NBS", "MAL"), c("RES", "MAL")),
label.y = c(7, 9.5),
label.y.npc = "bottom",
label = "p.signif",
method = "wilcox.test")Combine maternal (1a) serum, urine, and neonatal (1b) urine plots
p <- ggarrange(leucine, isoleucine, isobutyrate ,NAGP, glutamine, THB,
PAG, PCS, IS, PHPA, unkn, valine, aKIV, MM, creatinine, pb)
fig1 <- annotate_figure(p,
left = text_grob("Integral (a.u.)",
size = 10, rot = 90),
bottom = text_grob("Time point",
size = 10))
Table S5: Maternal metabolic changes depend on type of bariatric surgery
Relative concentration changes for each discriminatory metabolite were detailed at all time points for both malabsorptive and restrictive groups.
################## DEFINED VARIABLES ##################
# input dataframe has one row for each sample, one column for each metabolite (with concentrations or integrals)
input_data <- integrals
# metabolites of interest (match column names in input data)
metab_names <- c("PAG", "PCS", "IS", "PHPA", "unkn", "creatinine", "aKIV", "MM", "valine",
"isoleucine", "leucine", "isobutyrate", "glutamine", "THB", "NAGP")
# column name for time point
tp_col <- "Time_point_label"
# time points to include
time_points <- c("T1", "T2", "T3", "T4", "T5", "T6")
# column name for group
group_col <- "Group"
# case group name
case_names <- c("Restrictive", "Malabsorptive") # function only takes one input group, use for loop
# control group name
con_name <- "No Bariatric Surgery"
###########################################################Generate table: log2(Fold Change) was calculated where Fold Change (FC) = mean(case)/mean(control). P values are from Mann-Whitney U test.
univariate_metabolites <- function(input_data,
metab_names,
tp_col,
time_points,
group_col,
case_name,
con_name) {
# subset data to comparison groups
input_data <- subset(input_data, input_data[[group_col]] %in% c(case_name, con_name))
# create empty matrices to store results
log2FC <- as.data.frame(matrix(nrow = length(time_points), ncol = length(metab_names))) %>%
set_colnames(metab_names) %>%
set_rownames(time_points)
pval <- as.data.frame(matrix(nrow = length(time_points), ncol = length(metab_names))) %>%
set_colnames(metab_names) %>%
set_rownames(time_points)
# Calculate log2(FC) for each metabolite at time points T1 - T6
for (time in time_points){
data <- subset(input_data, input_data[[tp_col]] == time)
con <- subset(data, data[[group_col]] == con_name)
case <- subset(data, data[[group_col]] == case_name)
for (metab in metab_names){
log2fc <- round(log2(mean(case[,metab], na.rm = T)/mean(con[,metab], na.rm = T)), digits = 3)
log2FC[time,metab] <- log2fc
}}
# Calculate p value for each metabolite at time points T1 - T6
for (time in time_points){
for (metab in metab_names){
data <- subset(input_data,
input_data[[tp_col]] == time & input_data[[group_col]] %in% c(case_name, con_name))
form <- formula(paste(metab, "~", group_col))
pval[time,metab] <- wilcox.test(form, data, exact = FALSE)$p.value
}}
# adjust p values
log2FC.list <- data.frame(Time_point = rep(time_points, length(metab_names)),
bind_cols(gather(log2FC, "Metabolite", "log2FC"),
gather(pval, "Metab", "p.val")))
log2FC.list$p.adj.BH <- p.adjust(log2FC.list$p.val, method = "BH")
log2FC.list$Group <- case_name
return(log2FC.list[,-which(colnames(log2FC.list) == "Metab")])
}Generate table for each case group
tableS5 <- NULL
for (gr in case_names) {
idx <- univariate_metabolites(input_data,
metab_names,
tp_col,
time_points,
group_col,
case_name = gr,
con_name)
tableS5 <- rbind(tableS5, idx)
}Format table
# change p values to scientific notation
tableS5_formatted <- tableS5 %>%
mutate(P = formatC(tableS5$p.val, format = "e", digits = 2),
Padj = formatC(tableS5$p.adj.BH, format = "e", digits = 2))
# change non-significant p values to NS
tableS5_formatted$P[as.numeric(tableS5_formatted$P) > 0.05] <- "NS"
tableS5_formatted$Padj[as.numeric(tableS5_formatted$Padj) > 0.05] <- "NS"Figure S3: Maternal metabolic changes depend on type of bariatric surgery
Graphical representation of the changes
################## DEFINED VARIABLES ##################
input_data <- tableS5_formatted %>%
# label significant data
mutate(plot_sig = ifelse(Padj == "NS", NA, "*"),
Group_label = case_when(Group == "Malabsorptive" ~ "MAL",
Group == "Restrictive" ~ "RES"),
# Change names for plot titles
Metabolite = plyr::mapvalues(Metabolite,
from = c("THB", "NAGP", "unkn", "aKIV", "MM", "glutamine",
"creatinine", "isobutyrate", "leucine", "isoleucine",
"valine"),
to = c("D-beta-hydroxybutyrate", "GlycA",
"Unknown", "alpha-Ketoisovalerate", "Methylmalonate",
"Glutamine", "Creatinine", "Isobutyrate", "Leucine",
"Isoleucine", "Valine")),
# order time points
Time_point = factor(Time_point, levels = c("T6", "T5", "T4", "T3", "T2", "T1")),
# add which biofluid metabolites were identified in
biofluid = ifelse(Metabolite %in% c("PAG", "PCS", "IS", "PHPA", "Unknown", "Valine",
"Creatinine", "alpha-Ketoisovalerate", "Methylmalonate"),
"urine",
"serum"))
# column names
tp_col <- "Time_point"
metab_col <- "Metabolite"
s_type_col <- "biofluid"
group_col <- "Group_label"
plot_val <- "log2FC"
label_sig <- "plot_sig"
figure_labels <- list(serum = "a", urine = "b")
###########################################################Plot effect sizes and significance
for (s_type in names(figure_labels)) {
# subset to appropriate biofluid type
to_plot <- subset(input_data, input_data[[s_type_col]] == s_type)
# plot log2FC for both groups
p <- ggdotchart_not_ordered(to_plot,
x = tp_col,
xlab = "",
y = plot_val,
ylab = "",
rotate = TRUE,
color = group_col,
palette = unlist(color_pal),
dot.size = 1.5,
label = label_sig,
font.label = list(color = "black",
size = 10,
vjust = 0.72),
add = "segments",
add.param = list(color = "lightgray",
size = 0.4)) +
ylim(c(min(to_plot$log2FC), max(to_plot$log2FC))) +
geom_hline(yintercept = 0,
linetype = 2,
size = 0.4,
color = "lightgray") +
theme(legend.title = element_blank(),
legend.text=element_text(size = 6),
legend.key.size = unit(3, "mm"),
axis.text=element_text(size = 5),
axis.line = element_line(size = 0.4),
axis.ticks = element_line(size = 0.4)) +
rotate_x_text(55)
# separate plots for each metabolite
p <- facet(p,
facet.by = metab_col,
nrow = 2,
scales = "free_x",
panel.labs.background = list(fill = "ivory2",
color = "black"),
panel.labs.font = list(size = 4,
face ="bold"))
# add figure labels and axis annotations
p <- annotate_figure(p,
fig.lab.pos = "top.left",
fig.lab = figure_labels[[s_type]],
fig.lab.size = 10,
fig.lab.face = "bold",
left = text_grob("Time point",
size = 6,
rot = 90, vjust = 4),
bottom = text_grob("log2(Fold Change)",
size = 6, vjust = -4))
assign(paste0(s_type, "_dotplot"), p)
}Combine serum and urine plots
figS3 <- ggarrange(serum_dotplot, urine_dotplot,
ncol = 2,
widths = c(1, 1.5))
Figure S7: Relative concentrations of urinary host-microbial co-metabolites vary depending on bariatric surgery sub-type
Comparison of key discriminatory metabolites between study sub-groups.
################## DEFINED VARIABLES ##################
input_data <- integrals %>%
# separate restrictive group into band and sleeve
mutate(Group_label = case_when(Group_label == "NBS" ~ "NBS",
Group_label == "MAL" ~ "MAL",
Group_label == "RES" & Bariatric.surgery == "BAND" ~ "BAND",
Group_label == "RES" & Bariatric.surgery == "SLEEVE" ~ "SLEEVE")) %>%
# order sub-groups
mutate(Group_label = factor(Group_label, levels = c("NBS", "BAND", "SLEEVE", "MAL"))) %>%
# remove neonatal samples
filter(Time_point_label != "T7")
# column names
metab_names <- c("PAG", "PCS", "IS", "PHPA", "unkn")
tp_col <- "Time_point_label"
group_col <- "Group_label"
colors <- list(NBS = "purple", BAND = "coral2", SLEEVE = "coral2", MAL = "darkturquoise")
###########################################################For each metabolite, plot relative concentrations at each time point.
for (metab in metab_names) {
for (tp in unique(input_data[[tp_col]])) {
p <- ggboxplot(subset(input_data, input_data[[tp_col]] == tp),
size = 0.2,
group_col,
metab,
color = group_col,
palette = unlist(colors),
add = "jitter",
add.params = list(alpha = 0.5,
size = 0.3),
xlab = FALSE,
ylab = paste(metab, tp)) +
theme(legend.position = "None",
axis.text=element_text(size = 5),
axis.title = element_text(size = 6),
axis.line = element_line(size = 0.4),
axis.ticks = element_line(size = 0.4)) +
rotate_x_text(angle = 45)
p$layers[[1]]$geom_params$outlier.size <- 0.3 # change size of outlier points
assign(paste0(metab, "_", tp, "_boxplot"), p)
}
}Combine all plots together
figS7 <- ggarrange(PAG_T1_boxplot, PAG_T2_boxplot, PAG_T3_boxplot, PAG_T4_boxplot,
PAG_T5_boxplot, PAG_T6_boxplot, PCS_T1_boxplot, PCS_T2_boxplot,
PCS_T3_boxplot, PCS_T4_boxplot, PCS_T5_boxplot, PCS_T6_boxplot,
IS_T1_boxplot, IS_T2_boxplot, IS_T3_boxplot, IS_T4_boxplot,
IS_T5_boxplot, IS_T6_boxplot, PHPA_T1_boxplot, PHPA_T2_boxplot,
PHPA_T3_boxplot, PHPA_T4_boxplot, PHPA_T5_boxplot, PHPA_T6_boxplot,
unkn_T1_boxplot, unkn_T2_boxplot, unkn_T3_boxplot, unkn_T4_boxplot,
unkn_T5_boxplot, unkn_T6_boxplot)
Figure 2: Gut bacterial alterations in malabsorptive patients
################## DEFINED VARIABLES ##################
# input data is phyloseq object
input_data <- ps_M
# column names
tp_col <- "Time_point_label"
group_col <- "Group_label"
adiv_measure <- "Shannon"
bdiv_measure <- "bray"
ordination_meth <- "MDS"
set.seed(71)
###########################################################Calculate alpha-diversity
# normalize by rarefying to minimum sequencing depth
data <- input_data %>%
rarefy_even_depth(sample.size = min(sample_sums(input_data)), verbose = FALSE, replace = FALSE)
# calculate alpha diversity
alpha_div <- estimate_richness(data, split = TRUE, measures = adiv_measure)
# add variables needed for plot
alpha_div$Time_point <- sample_data(data)[[tp_col]]
alpha_div$Group <- sample_data(data)[[group_col]]Calculate beta-diversity
# use log transformation normalization to overcome differences in sequencing depth
data <- input_data %>%
transform_sample_counts(function(x) {log(x+1)})
bdiv <- phyloseq::distance(data, bdiv_measure)
MDS <- ordinate(data, ordination_meth, bdiv)
# extract % variance explained for axis labels
ord <- plot_ordination(data, MDS)[["labels"]] (2a) Comparison of Shannon Index between malabsorptive and control groups
am <- facet(ggboxplot(alpha_div,
x = "Group",
y = adiv_measure,
color = "Group",
add = "jitter",
add.params = list(alpha = 0.5),
size = 0.5,
palette = unlist(color_pal),
legend = "none",
ylim = c(3.3,5.2),
xlab = FALSE,
ylab = "Shannon's Diversity Index (H)") +
theme(axis.text = element_text(size = 6),
axis.title = element_text(size = 6)) +
# add significance
stat_compare_means(comparisons = list(c("NBS", "MAL")),
label = "p.signif",
method = "wilcox.test",
label.y = 5.1,
hide.ns = TRUE),
facet.by = "Time_point",
panel.labs.background = list(fill = "ivory2", color = "black"),
panel.labs.font = list(size = 7, face ="bold"))(2b) Bray-Curtis dissimilarity with PCoA ordination
# plot PCoA
to_plot <- plot_ordination(data, MDS, justDF = TRUE)
bm <- ggscatter(to_plot,
x = "Axis.1",
y = "Axis.2",
color = "Group_label",
alpha = 0.6,
ggtheme = theme_bw(),
shape = "Time_point_label",
size = 1.5,
ellipse = TRUE,
ellipse.level = 0.95,
ellipse.type = "norm",
ellipse.alpha = 0) +
scale_colour_manual(values = unlist(color_pal)) +
labs(x = str_replace(ord$x, "Axis.1 ", "PC1"),
y = str_replace(ord$y, "Axis.2 ", "PC2")) +
theme(legend.position = "none",
axis.title =element_text(size=6),
axis.text = element_text(size = 6))
# permanova test
adonis_res <- adonis(bdiv ~ Group, data.frame(sample_data(data)))
# add permanova p value
bm <- bm +
annotate("text",
size = 2,
x = 0.3,
y = 0.32,
label = paste("P = ", round(adonis_res$aov.tab$`Pr(>F)`[1], digits = 4)))
# put 2a and 2b together
plot1 <- ggarrange(am, bm, labels = c("a", "b"), widths = c(2,1))(2c) Differential abundance (malabsorptive vs control) at each taxonomic level. Effect sizes are log2(FC) and P values are from Mann-Whitney U test.
################## DEFINED VARIABLES ##################
# input data is phyloseq object
data <- ps_M
tp_col <- "Time_point_label"
time_points <- c("T1", "T2", "T4")
group_col <- "Group_label"
case_name <- "MAL"
con_name <- "NBS"
# 16S data transformation parameters
# taxa without at least min_counts in at least prev_filt (%) of samples will be removed
min_counts <- 5
prev_filt <- 0.1
###########################################################Generate differential abundance table (Table S6)
MWU <- NULL
for (rank in c("Phylum", "Class", "Order", "Family", "Genus")){
for (time in time_points){
ps_obj <- data %>%
tax_glom(rank) %>%
# prevalence filter
filter_taxa(function(x) sum(x > min_counts) > (prev_filt*length(x)), TRUE) %>%
# normalize by total sum scaling
transform_sample_counts(function(x) x/sum(x)) %>%
# subset to time point
subset_samples(Time_point_label == time)
tax <- data.frame(as(tax_table(ps_obj), "matrix"))
abund <- data.frame(as(otu_table(ps_obj), "matrix")) %>%
# change column names to names at current rank
set_colnames(gsub("/", ".", tax[,which(colnames(tax)==rank)]))
# add group column
abund$Group <- sample_data(ps_obj)[[group_col]]
# test differences between groups for every taxon
for (i in c(1:(ncol(abund)-1))) {
taxa <- colnames(abund)[1:(ncol(abund)-1)]
form <- formula(paste(taxa[i], "~ Group"))
mw <- wilcox.test(form, abund, exact = FALSE)
idx <- tibble(Rank = rank,
Time_point = time,
Taxon = taxa[i],
Mean_rel_abund_case = mean(subset(abund, abund$Group == case_name)[,i]),
Mean_rel_abund_cont = mean(subset(abund, abund$Group == con_name)[,i]),
pval = mw$p.value,
log2FC = log2(mean(subset(abund, abund$Group == case_name)[,i])/
mean(subset(abund, abund$Group == con_name)[,i]))
)
MWU <- rbind(MWU, idx)
}
}
}
# multiple hypothesis testing correction
MWU$p.adj.BH <- p.adjust(MWU$pval, method = "BH")
# filter to significant taxa; excluding taxa that are not observed in a group (zero counts)
MWU_filt <- subset(MWU, MWU$p.adj.BH < 0.05 & abs(MWU$log2FC) != Inf)Barplots for each rank
# color related taxa
palette <- list(Proteobacteria = "cyan4",
Bacilli = "khaki4",
Actinomycetales = "chocolate3",
Anaerostipes = "thistle4",
NS = "grey")
diff_abund <- MWU %>%
# add x-axis plot label
mutate(Label = str_replace(paste0(Taxon, "_", Time_point),
"Escherichia\\.", "Escherichia/"),
# group taxa for coloring
Taxa_group = case_when(p.adj.BH > 0.05 ~ "NS",
grepl("Proteobact|Gammaprot|Enterobacteria|Escherichia|Pasteurell|Haemophil",
Taxon) ~ "Proteobacteria",
Taxon %in% c("Micrococcaceae", "Rothia", "Actinomycetales") ~ "Actinomycetales",
grepl("Bacill|Enteroc|Strepto|Lactobacill|Carnobact|Granulicat",
Taxon) ~ "Bacilli",
grepl("Bacteroid|Butyricimonas", Taxon) ~ "Bacteroidetes",
Taxon == "Anaerostipes" ~ "Anaerostipes",
grepl("Holdeman|Solobact", Taxon) ~ "Erysipelotrichaceae",
grepl("Veillonella|Acidaminococcus", Taxon) ~ "Negativicutes",
grepl("Clostrid", Taxon) ~ "Clostridia"))
# factor for correct ordering on plot
diff_abund$Rank <- factor(diff_abund$Rank, levels = c("Phylum", "Class", "Order", "Family", "Genus"))
diff_abund$Taxa_group <- factor(diff_abund$Taxa_group, levels = c("Proteobacteria",
"Bacilli",
"Bacteroidetes",
"Clostridia",
"Negativicutes",
"Actinomycetales",
"Erysipelotrichaceae",
"Anaerostipes",
"NS"))
# remove taxa only significant at one time point
MWU_plot <- subset(diff_abund, diff_abund$Taxon %in%
names(which(table(MWU_filt$Taxon) > 1)))
# top barplots
p <- ggbarplot(subset(MWU_plot, MWU_plot$Rank %in% c("Phylum", "Class", "Order")),
x = "Label",
y = "log2FC",
lab.size = 4,
fill = "Taxa_group",
color = "white",
palette = unlist(palette),
ylab = "log2(Fold Change)",
lab.vjust = .5,
xlab = FALSE,
order = sort(MWU_plot$Label)) +
theme(axis.title = element_text(size=6),
axis.text = element_text(size = 6),
legend.position = "none") +
rotate_x_text(55)
p <- facet(p,
facet.by = "Rank",
scales = "free_x",
nrow = 1,
space = "free_x",
panel.labs.background = list(fill = "ivory2", color = "black"),
panel.labs.font = list(size = 8, face ="bold"))
# bottom barplots
q <- ggbarplot(subset(MWU_plot, MWU_plot$Rank %in% c("Family", "Genus")),
x = "Label",
y = "log2FC",
lab.size = 4,
fill = "Taxa_group",
color = "white",
palette = unlist(palette),
ylab = "log2(Fold Change)",
lab.vjust = .5,
xlab = FALSE,
order = sort(MWU_plot$Label)) +
theme(axis.title = element_text(size=6),
axis.text = element_text(size = 6),
legend.position = "right",
legend.text = element_text(size = 6),
legend.title = element_text(size = 8),
legend.key.size = unit(4, "mm")) +
rotate_x_text(55) +
geom_hline(yintercept = 0, linetype="dashed", size = 0.4)
q <- facet(q,
facet.by = "Rank",
scales = "free_x",
nrow = 1,
space = "free_x",
panel.labs.background = list(fill = "ivory2", color = "black"),
panel.labs.font = list(size = 7, face ="bold"))
# combine barplots
plot2 <- ggarrange(p, q, nrow = 2)Combine (2c) with (2a) and (2b)
fig2 <- ggarrange(plot1, plot2,
nrow = 2,
heights = c(1.2,2),
labels = c("", "c"))
Table S7: Differentially abundant amplicon sequence variants
Differential abundance of amplicon sequence variants was assessed by DESeq2. Taxonomic assignments at species level were compared between SILVA and RDP databases.
################## DEFINED VARIABLES ##################
input_data <- merge_samples(ps_M, "Study.no")
# fix group labels - merging samples changes labels to numbers
sample_data(input_data)$Group <- str_replace_all(sample_data(input_data)$Group,
c("1" = "Control",
"2" = "Malabsorptive"))
sample_data(input_data)$Group <- factor(sample_data(input_data)$Group, levels = c("Control", "Malabsorptive"))
# 16S data transformation parameters
# taxa without at least min_counts in at least prev_filt (%) of samples will be removed
min_counts <- 5
prev_filt <- 0.1
alpha <- 0.05
gen <- c("Enterococcus", "Escherichia/Shigella", "Streptococcus", "Rothia")
###########################################################Make table with differential abundance and species assignments
# prevalence filter
ps_trim <- input_data %>%
filter_taxa(function(x) sum(x > min_counts)> (prev_filt*length(x)), TRUE)
# run DESeq2
DDS <- phyloseq_to_deseq2(ps_trim, ~ Group)
DDS <- estimateSizeFactors(DDS, type = "poscounts")
DDS <- estimateDispersions(DDS, fitType = "local")
DDS <- DESeq(DDS, fitType = "local")
# results
res <- results(DDS)
res <- res[order(res$padj, na.last=NA), ]
sigtab <- res[(res$padj < alpha), ]
sigtab <- cbind(as(sigtab, "data.frame"), as(tax_table(ps_trim)[rownames(sigtab), ], "matrix"))
# keep only genera of interest
sigtab <- subset(sigtab, sigtab$Genus %in% gen)
keep <- which(row.names(rdp.species) %in% row.names(sigtab))
rdp.species <- data.frame(rdp.species)
rdp.species$seq <- row.names(rdp.species)
silva.species <- data.frame(silva.species)
silva.species$seq <- row.names(silva.species)
# combine RDP and SILVA species assignments
species_assign <- merge(rdp.species[keep, c(6:8)], silva.species[keep, c(6:8)], by = "seq")
# combine DESeq2 results with species assignments
sigtab$seq <- row.names(sigtab)
species_assign <- merge(sigtab[,c(1:6,13)], species_assign, by = "seq")Figure 3: Integration of serum, urine and faecal profiles
Relative concentrations of key discriminatory metabolites were integrated with genera abundances using “DIABLO”, a method implemented in mixOmics.
################## DEFINED VARIABLES ##################
# input data is phyloseq object
input_MB <- ps_M %>%
tax_glom("Genus") %>%
transform_sample_counts(function(x) {log(x+1)})
# remove neonatal samples & subset to individuals with microbiome data
input_metab <- integrals %>%
filter(Time_point != 7)
input_metab <- left_join(data.frame(Patient.TP = sample_names(input_MB)),
input_metab,
by = "Patient.TP")
S_metab_names <- c("leucine", "isoleucine", "isobutyrate", "THB")
U_metab_names <- c("PAG", "PCS", "IS", "PHPA", "unkn", "aKIV", "creatinine")
# number of genera to keep in model
n_gen <- 10
###########################################################Prepare datasets
tax <- as(tax_table(input_MB), "matrix") %>%
data.frame(stringsAsFactors = F)
F_abund_mat <- as(otu_table(input_MB), "matrix") %>%
data.frame() %>%
set_colnames(tax$Genus)
S_NMR_mat <- input_metab %>%
select(one_of(S_metab_names)) %>%
set_rownames(input_metab$Patient.TP)
U_NMR_mat <- input_metab %>%
select(one_of(U_metab_names)) %>%
set_rownames(input_metab$Patient.TP)Build model
X <- list(Urine = U_NMR_mat,
Serum = S_NMR_mat,
Faeces = F_abund_mat)
Y <- input_metab[, "Group_label"]
# only model top n taxa for each component
list.keepX <- list(Urine = ncol(U_NMR_mat), Serum = ncol(S_NMR_mat), Faeces = c(n_gen, n_gen))
# multi-block pls-da
m <- block.splsda(X, Y,
keepX = list.keepX,
near.zero.var = TRUE)Plot sample scores for each dataset. Separation between classes is on Component 1.
ggsave(plotIndiv(m,
legend = TRUE,
X.label = "Component 1",
Y.label = "Component 2",
legend.title = '',
style = "lattice",
ind.names = FALSE,
col.per.group = unlist(color_pal)[-2],
ellipse = TRUE,
legend.position = "top",
pch = 20, alpha = 0.7,
size.axis = 0.5, size.xlabel = 0.9, size.ylabel = 0.9,
size.legend = 0.8) +
theme(legend.box.background = element_rect()),
filename = file.path(save_dir, "figure3a_integration_scores.pdf"),
height = 3, width = 6)Plot correlations between variables and components highlighting variables with high correlation on either component.
var_plot <- plotVar(m, plot = F) %>%
mutate(Plot_label = plyr::mapvalues(names,
from = c("THB", "unkn", "aKIV", "creatinine",
"isobutyrate", "leucine", "isoleucine"),
to = c("D-beta-HB", "Unknown",
"alpha-Ketoisovalerate", "Creatinine",
"Isobutyrate", "Leucine", "Isoleucine")),
Plot_label = case_when(abs(x) > 0.5 | abs(y) >= 0.5 ~ Plot_label),
Plot_alpha = ifelse(is.na(Plot_label), "|r|<0.5", "|r|>=0.5"))
ggsave(ggscatter(var_plot, x = "x", y = "y",
color = "Block", palette = c("darkolivegreen", "darkgoldenrod3", "rosybrown3"),
label = "Plot_label", repel = TRUE,
font.label = c(5, "plain"),
label.rectangle = FALSE,
alpha = "Plot_alpha",
xlab = "Component 1", ylab = "Component 2",
show.legend.text = FALSE) +
xlim(c(-1, 1)) +
ylim(c(-1,1)) +
geom_hline(yintercept = 0,
linetype = 2,
size = 0.4,
color = "lightgray") +
geom_vline(xintercept = 0,
linetype = 2,
size = 0.4,
color = "lightgray") +
theme(legend.title = element_blank(),
axis.text = element_text(size = 5),
axis.title = element_text(size = 8),
legend.text = element_text(size = 5)),
filename = file.path(save_dir, "figure3b_integration_variables.pdf"),
height = 3.5, width = 3.5) Plot correlations between datasets for Component 1.
ggsave(circosPlot(m,
cutoff=0.5,
comp = 1,
color.cor = c("red", "blue"),
color.blocks = c("rosybrown3", "darkgoldenrod3", "darkolivegreen"),
showIntraLinks = FALSE,
legend = TRUE,
line = FALSE,
size.labels = 0.01,
size.variables = 0.5,
var.names = list(Urine = c("PAG","PCS","IS","PHPA","Unknown","alpha-KIV","Creatinine"),
Serum = c("Leucine", "Isoleucine", "Isobutyrate", "D-beta-HB"),
Faeces = m$names$colnames$Faeces),
size.legend = 0.5) +
theme(legend.box.background = element_rect()),
filename = file.path(save_dir, "figure3c_integration_circos.pdf"),
height = 4, width = 4.5)
Figure 3 (combined)
Figure 4: Relative concentrations of metabolites correlate with maternal insulin resistance and fetal/birth weight
Correlations (Spearman) were calculated between metabolite relative concentrations and clinical measures. Netowrk file was generated for Cytoscape input.
################## DEFINED VARIABLES ##################
time_points <- 1:6
input_data <- subset(integrals, integrals$Time_point %in% time_points)
tp_col <- "Time_point_label"
# column names
metab_names <- c("PAG", "PCS", "IS", "isoleucine", "leucine", "isobutyrate")
clin_varT1 <- c("Fast Maternal Insulin (microU/ml)",
"Maternal HOMA-IR",
"Fast Maternal Gluc (mmol/L)",
"GA..days.",
"BMI.1",
"Age")
clin_varT2 <- c("Fast Maternal Insulin (microU/ml)",
"Maternal HOMA-IR",
"Fast Maternal Gluc (mmol/L)",
"GA..days.",
"BMI2",
"EFW2-centile",
"Age")
clin_varT3 <- c("Fast Maternal Insulin (microU/ml)",
"Maternal HOMA-IR",
"Fast Maternal Gluc (mmol/L)",
"GA..days.",
"BMI3",
"Age")
clin_varT4 <- c("Fast Maternal Insulin (microU/ml)",
"Maternal HOMA-IR",
"Fast Maternal Gluc (mmol/L)",
"GA..days.",
"BMI4",
"EFW4-centile",
"Age")
clin_varT5 <- c("Fast Maternal Insulin (microU/ml)",
"Maternal HOMA-IR",
"Fast Maternal Gluc (mmol/L)",
"GA..days.",
"BMI5",
"EFW5-centile",
"Age")
clin_varT6 <- c("Fast Maternal Insulin (microU/ml)",
"Maternal HOMA-IR",
"Fast Maternal Gluc (mmol/L)",
"GA..days.",
"BMI6",
"BW.centile",
"Age")
########################################################### Generate network file
# use T5 BMI for T6 (visits are couple weeks apart, BMI not measured at T6)
input_data$BMI6 <- input_data$BMI5
# format data for correlation at each time point
for (time in unique(input_data[[tp_col]])) {
idx <- subset(input_data, input_data[[tp_col]] == time)
# keep only columns to correlate
idx <- idx[,which(colnames(idx) %in% c(metab_names, get(grep(paste0("clin_var", time), ls(), value = T))))]
# fix column names so they all match
colnames(idx) <- str_replace_all(colnames(idx), c("BMI.*" = "BMI", ".*centile" = "EFW.BW"))
if (!"EFW.BW" %in% colnames(idx)) { idx$EFW.BW <- NA }
# tidy column names
colnames(idx) <- make.names(colnames(idx))
# arrange columns in the same order
idx <- idx[,c(metab_names,
"Age",
"BMI",
"GA..days.",
"Fast.Maternal.Insulin..microU.ml.",
"Fast.Maternal.Gluc..mmol.L.",
"Maternal.HOMA.IR",
"EFW.BW")]
# make sure all columns are numeric
idx <- sapply(idx, as.numeric)
assign(paste(time), idx)
}
# remove confounders
for (time in unique(input_data[[tp_col]])) {
idx <- get(paste(time))
assign(paste0(time, "_to_corr"), idx[,-c(which(colnames(idx) %in% c("Age", "BMI")))])
}
# correlation at each time point
corr_all <- NULL
corr_pval <- NULL
for (cor in time_points) {
idx <- get(grep(paste0(cor,"_to_corr"), ls(), value = T))
rcorr <- rcorr(idx, type = "spearman")
coef <- round(rcorr$r,2)
coef <- coef[c(which(rownames(coef) %in% metab_names)), c(which(!colnames(coef) %in% metab_names))]
row.names(coef) <- paste0(metab_names, "_T", cor)
pval <- rcorr$P
pval <- pval[c(which(rownames(pval) %in% metab_names)), c(which(!colnames(pval) %in% metab_names))]
row.names(pval) <- paste0(metab_names, "_T", cor)
corr_all <- rbind(corr_all, coef)
corr_pval <- rbind(corr_pval, pval)
}
# change to long format and adjust p values
corr_all <- melt(corr_all, value.name = "spearman")
corr_all <- subset(corr_all, !is.na(corr_all$spearman))
corr_pval <- melt(corr_pval, value.name = "p.val")
corr_pval <- subset(corr_pval, !is.na(corr_pval$p.val))
corr_pval$p.adj.BH <- p.adjust(corr_pval$p.val, method = "BH")
corr_metaVmetab <- cbind(corr_all, corr_pval[,c(3:4)])
corr_metaVmetab_sig <- subset(corr_metaVmetab, corr_metaVmetab$p.adj.BH < 0.05)
# convert to graph for cytoscape visualisation
cytoscape <- corr_metaVmetab_sig[,c(1:3)] %>%
mutate(Strength = case_when(abs(spearman) < 0.4 ~ "0.3-0.4",
abs(spearman) >= 0.4 ~ "0.4-0.5"),
Direction = case_when(spearman > 0 ~ "pos",
spearman < 0 ~ "neg")) %>%
rename(from = Var1, to = Var2, weight = spearman)
net <- graph.data.frame(cytoscape, directed = FALSE)
write_graph(net, file.path(save_dir, "cormat_cytoscape.gml"), format = "gml")
Figure 4 (generated with Cytoscape)
Table S8: Relative concentrations of metabolites correlate with maternal insulin resistance and fetal/birth weight
Includes partial correlations to adjust for the effect of maternal age and BMI as well as any effect of maternal insulin resistance on the baby’s weight.
corr_metaVmetab_sig$partial <- NA
corr_metaVmetab_sig$part.pval <- NA
for (part_cor in c(1:nrow(corr_metaVmetab_sig))) {
var1 <- gsub("_T.*", "", corr_metaVmetab_sig[part_cor,1])
var2 <- corr_metaVmetab_sig[part_cor,2]
idx <- get(paste0(gsub(".*_", "", corr_metaVmetab_sig[part_cor,1])))
if (var2 == "EFW.BW") {
conf <- which(colnames(idx) %in% c("Age", "BMI", "Maternal.HOMA.IR"))
# remove any rows with missing data
idx <- subset(idx,
!is.na(idx[,which(colnames(idx)==var1)]) &
!is.na(idx[,which(colnames(idx)==var2)]) &
!is.na(idx[,which(colnames(idx)=="Maternal.HOMA.IR")]) &
!is.na(idx[,which(colnames(idx)=="BMI")]) &
!is.na(idx[,which(colnames(idx)=="Age")]))
} else {
conf <- which(colnames(idx) %in% c("Age", "BMI"))
# remove any rows with missing data
idx <- subset(idx,
!is.na(idx[,which(colnames(idx)==var1)]) &
!is.na(idx[,which(colnames(idx)==var2)]) &
!is.na(idx[,which(colnames(idx)=="BMI")]) &
!is.na(idx[,which(colnames(idx)=="Age")]))
}
# partial correlations
part_corr <- pcor.test(idx[,which(colnames(idx)==var1)],
idx[,which(colnames(idx)==var2)],
idx[,conf],
method = "spearman")
corr_metaVmetab_sig$partial[part_cor] <- part_corr$estimate
corr_metaVmetab_sig$part.pval[part_cor] <- part_corr$p.value
corr_metaVmetab_sig$part.pval.adj <- p.adjust(corr_metaVmetab_sig$part.pval, method = "BH")
}
for (col in c("p.val","p.adj.BH","part.pval","part.pval.adj")) {
corr_metaVmetab_sig[as.numeric(corr_metaVmetab_sig[[col]]) > 0.05, col] <- "NS"
}Figure S8: Key metabolites are not associated with percentage of weight lost or length of time since surgery in malabsorptive patients
to_cor <- integrals %>%
filter(Group_label == "MAL",
Time_point != 7)
for (variab in c("perc_wl", "Months_betw_op_concep")) {
for (metab in c("PAG", "PCS", "IS")) {
p <- ggscatter(to_cor,
x = variab,
xlab = str_replace_all(variab,
c("perc_wl" = "Post-surgery weight loss (%)",
"Months_betw_op_concep" =
"Time between operation and conception (months)")),
y = metab,
ylab = paste(metab, "(a.u.)"),
facet.by = "Time_point_label",
alpha = 0.7,
size = 1,
color = "darkturquoise",
cor.coef = TRUE,
cor.coeff.args = list(method = "spearman"),
cor.coef.size = 1.5) +
theme(axis.text = element_text(size = 6),
axis.title = element_text(size = 7))
assign(paste0(metab, "_", variab), p)
}
}
p <- ggarrange(PAG_perc_wl, PCS_perc_wl, IS_perc_wl,
PAG_Months_betw_op_concep, PCS_Months_betw_op_concep, IS_Months_betw_op_concep)
Session Info
sessionInfo()## R version 3.5.0 (2018-04-23)
## Platform: x86_64-apple-darwin15.6.0 (64-bit)
## Running under: macOS 10.14.6
##
## Matrix products: default
## BLAS: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRblas.0.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRlapack.dylib
##
## locale:
## [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8
##
## attached base packages:
## [1] parallel stats4 stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] plotly_4.8.0 reshape2_1.4.3
## [3] ppcor_1.1 igraph_1.2.4
## [5] Hmisc_4.2-0 Formula_1.2-3
## [7] survival_2.44-1.1 mixOmics_6.6.2
## [9] MASS_7.3-51.4 DESeq2_1.22.2
## [11] SummarizedExperiment_1.12.0 DelayedArray_0.8.0
## [13] BiocParallel_1.16.6 matrixStats_0.54.0
## [15] Biobase_2.42.0 GenomicRanges_1.34.0
## [17] GenomeInfoDb_1.18.2 IRanges_2.16.0
## [19] S4Vectors_0.20.1 BiocGenerics_0.28.0
## [21] vegan_2.5-4 lattice_0.20-38
## [23] permute_0.9-5 ggpubr_0.2
## [25] magrittr_1.5 phyloseq_1.26.1
## [27] santaR_1.0 forcats_0.4.0
## [29] stringr_1.4.0 dplyr_0.8.0.1
## [31] purrr_0.3.2 readr_1.3.1
## [33] tidyr_0.8.3 tibble_2.1.1
## [35] ggplot2_3.1.1 tidyverse_1.2.1
##
## loaded via a namespace (and not attached):
## [1] readxl_1.3.1 backports_1.1.3 plyr_1.8.4
## [4] lazyeval_0.2.2 splines_3.5.0 crosstalk_1.0.0
## [7] digest_0.6.18 foreach_1.4.4 htmltools_0.3.6
## [10] checkmate_1.9.1 memoise_1.1.0 cluster_2.0.8
## [13] doParallel_1.0.14 Biostrings_2.50.2 annotate_1.60.1
## [16] modelr_0.1.4 rARPACK_0.11-0 colorspace_1.4-1
## [19] ggrepel_0.8.0 blob_1.1.1 rvest_0.3.2
## [22] haven_2.1.0 xfun_0.6 crayon_1.3.4
## [25] RCurl_1.95-4.12 jsonlite_1.6 genefilter_1.64.0
## [28] iterators_1.0.10 ape_5.3 glue_1.3.1
## [31] gtable_0.3.0 zlibbioc_1.28.0 XVector_0.22.0
## [34] Rhdf5lib_1.4.3 scales_1.0.0 DBI_1.0.0
## [37] Rcpp_1.0.1 viridisLite_0.3.0 xtable_1.8-3
## [40] htmlTable_1.13.1 foreign_0.8-71 bit_1.1-14
## [43] htmlwidgets_1.3 httr_1.4.0 RColorBrewer_1.1-2
## [46] acepack_1.4.1 pkgconfig_2.0.2 XML_3.98-1.19
## [49] nnet_7.3-12 locfit_1.5-9.1 labeling_0.3
## [52] tidyselect_0.2.5 rlang_0.3.4 later_0.8.0
## [55] AnnotationDbi_1.44.0 munsell_0.5.0 cellranger_1.1.0
## [58] tools_3.5.0 cli_1.1.0 generics_0.0.2
## [61] RSQLite_2.1.1 ade4_1.7-13 broom_0.5.2
## [64] evaluate_0.13 biomformat_1.10.1 yaml_2.2.0
## [67] knitr_1.22 bit64_0.9-7 nlme_3.1-138
## [70] mime_0.6 xml2_1.2.0 compiler_3.5.0
## [73] shinythemes_1.1.2 rstudioapi_0.10 ggsignif_0.5.0
## [76] geneplotter_1.60.0 stringi_1.4.3 RSpectra_0.14-0
## [79] Matrix_1.2-17 multtest_2.38.0 pillar_1.3.1
## [82] cowplot_0.9.4 data.table_1.12.2 bitops_1.0-6
## [85] corpcor_1.6.9 httpuv_1.5.1 R6_2.4.0
## [88] latticeExtra_0.6-28 promises_1.0.1 gridExtra_2.3
## [91] codetools_0.2-16 assertthat_0.2.1 rhdf5_2.26.2
## [94] withr_2.1.2 GenomeInfoDbData_1.2.0 mgcv_1.8-28
## [97] hms_0.4.2 grid_3.5.0 rpart_4.1-13
## [100] rmarkdown_1.12 shiny_1.3.0 lubridate_1.7.4
## [103] base64enc_0.1-3 ellipse_0.4.1